Postmating isolation and evolutionary relationships among Fejervarya species from Lesser Sunda, Indonesia and other Asian countries revealed by crossing experiments and mtDNA Cytb sequence analyses

Abstract To evaluate the degree of postmating isolation and the evolutionary relationships among frog species in the genus Fejervarya from Indonesia (Lesser Sunda), Bangladesh, China, and Japan, crossing experiments and molecular phylogenetic analyses were carried out. Crossing experiments revealed that reciprocal hybrids among F. iskandari, F. verruculosa, and F. sp. large type and between F. multistriata and F. kawamurai are viable through metamorphosis, while those between the F. iskandari group and F. limnocharis group were completely or partially inviable at the tadpole stage and those between Southeast Asian and South Asian Fejervarya groups were completely inviable at the embryonic stage. The mature reciprocal hybrids between F. iskandari and F. verruculosa from Lesser Sunda, Indonesia, showed some degree of abnormality in spermatogenesis. In phylogenetic analyses based on mtDNA Cytb sequences, F. iskandari formed a sister clade with F. verruculosa from Lesser Sunda, Indonesia, with 8.1% sequence divergence. F. multistriata from China formed a clade with populations of F. limnocharis in Thailand, Malaysia, and Indonesia (topotype), and these taxa showed sister relationships to F. kawamurai from Japan with 8.9% sequence divergence. Fejervarya sp. small type from Bangladesh formed a clade with the other South Asian members of the Fejervarya group and formed a sister clade with the Southeast Asian Fejervarya group, with 23.1% sequence divergence in the Cytb gene. These results showed that the degree of postmating isolation reflects molecular phylogenetic relationships and that F. iskandari and F. verruculosa from Indonesia (Lesser Sunda) are reproductively isolated by abnormalities in spermatogenesis and show genetic differentiation.


| INTRODUC TI ON
A species is an indispensable entity in biological science (Claridge et al., 1997;Ereshefsky, 1992;Mayr, 1982), and recent approaches for species identification are debated among scientists and yield conflicting results for targeted taxa; thus, the perception of what constitutes a species continues to be a challenging dilemma (de Queiroz, 1998;Dobzhansky, 1976). Although Mayden (1997Mayden ( , 1999 drafted around 24 titled species concepts, the biological species concept remains the most central definition of the qualities of distinct species. If two taxa are reproductively isolated and cannot interbreed, they should be recognized as distinct species. It is possible that species are first isolated from each other, either completely or incompletely, by gametic isolation. When gametic isolation is incomplete, species are completely or incompletely isolated by hybrid inviability, and when hybrid inviability is incomplete, species are completely isolated by hybrid sterility (Sumida et al., 2003).
The most widely distributed frogs in Asia are Fejervarya limnocharis (Annandale, 1918). Recently, the species was transferred from the genus Rana to Fejervarya (Dubois & Ohler, 2000). This species is widely distributed in South to Southeast Asia, including many islands in Indonesia and Malaysia, northern, central, southern, and southwestern China, and Western Japan. A lack of diagnostic morphological characters makes it difficult for researchers to clearly separate these frogs, which are collectively referred to as the Fejervarya limnocharis complex. Many scientists have focused on the systematics of the Fejervarya limnocharis species complex. This species was first described in Java, Indonesia (Gravenhorst, 1829;Wiegmann, 1834).
To date, 14 species have been listed in the genus Fejervarya (Frost, 2021), and several species have been described in this species complex. Additional analytical work and more extensive studies are needed to delimit all distinct species within the F. limnocharis complex along with their geographic ranges. Djong et al. (2007) argued that the F. limnocharis complex can be divided into two subgroups (i.e., the F. limnocharis group and F. iskandari group). In this classification, the F. limnocharis group includes the topotypic F. limnocharis (Java specimen, Indonesia) and populations in Malaysia and Japan, whereas the F. iskandari group consists of F. iskandari and populations from Thailand and Bangladesh.
Analyzing and quantifying biological heterogeneity is a substantial scientific endeavor (Rivera-Correa et al., 2021). Only a tiny portion of the species on Earth has been explored, described, and identified, and we are far from generating an all-inclusive inventory of the biosphere (Moura & Jetz, 2021;Wheeler et al., 2012).
Importantly, Fejervarya specimens from Kupang, Ende and Maumere, Lesser Sunda have not been explored by artificial breeding to clearly determine whether the population belongs to the F. iskandari group or not. Therefore, in the current study, we examined specimens from the Lesser Sunda Islands to determine their phylogenetic affinities and to determine levels of reproductive isolation (if any) between the population and other known species from mainland Asia.
In the present study, a crossing experiment with six species was performed to clarify the reproductive isolating mechanisms. In addition, mtDNA gene sequence analyses of 27 frogs belonging to the genus Fejervarya from Indonesia, Bangladesh, Japan, and China were performed to determine the evolutionary relationships and levels of divergence within the F. limnocharis complex.

| Crossing experiments
Crossing experiments were performed by artificial insemination (Kawamura et al., 1980)

| Histological and spermatogenesis observations
Testes of the mature hybrids and control frogs were used for histological and spermatogenesis examinations. For each individual, one testis was fixed in Navashin's solution, sectioned at 10 μm, and stained with Heidenhain's iron hematoxylin for histological analyses.
The other testis was used for chromosome preparations. Meiotic chromosomes were prepared according to the procedure described by Schmid et al. (1979) with minor alterations. The chromosomes were stained with a 2% Giemsa solution for 5 min. The chromosome analysis was performed using only diploid cells at the diakinesis and metaphase stages of the first reduction division, as bivalent and univalent chromosomes could be conveniently differentiated from each other. Bivalent chromosomes were similar to normal chromosomes, with a thick, symmetrical form, whereas univalent chromosomes were indistinguishable from abnormal chromosomes, which were asymmetric and lean (Kawamura et al., 1980(Kawamura et al., , 1981Kuramoto, 1983;Sumida et al., 2003).  Alam et al., 2010). PCR amplification and sequencing were performed using the primers Fow-1-1 and Rev-1  to obtain a fragment of ca. 667 bp. The resultant nucleotide sequences were aligned using the ClustalW program (Thompson et al., 1994). A phylogeny was constructed using the maximum likelihood (ML) method implemented in Treefinder (Jobb, 2008), and branch support was evaluated by a

| Observations of the testes and spermatogenesis
To further clarify the relationships among these species, the inner structures of the testes from mature male hybrids between F. verruculosa and F. iskandari and the controls were quantified by histological analyses and observations of spermatogenesis ( Figures 5   and 6). The inner arrangements of the testes of control males were completely normal; seminiferous tubules were filled with tight bundles of normal spermatozoa ( Figure 5a). In contrast, the testes of the hybrids were slightly abnormal, with seminiferous tubules containing pycnotic nuclei in addition to normal bundles of spermatozoa ( Figure 5b).
In controls, 26 meiotic spreads were interpreted from two males, all of which consisted of 13 bivalents and no univalents (Table 3, Figure 6a). In the hybrids, of the 41 meiotic spreads interpreted from four hybrid males, one (2.4%) consisted of 13 bivalents and no univalents, 16 (39.0%) consisted of 12 bivalents and 2 univalents, and 24 (58.5%) consisted of 11 bivalents and four univalents (Table 3, Figure 6b). The mean number of univalents per spermatocyte was 3.12 and the proportion of univalents among all chromosomes was 12.0% (Table 3). Ring-shaped bivalents outnumbered rod-shaped bivalents substantially in the controls, whereas relative frequencies of ring-shaped bivalents decreased and rod-shaped bivalents increased in the hybrids in both the large and small chromosomes (Table 4, Figure 6). In total, 104 (80.0%) and 146 (70.2%) large and small bivalent chromosomes were ringshaped in the controls, respectively, whereas 131 (68.2%) and 161 (49.7%) large and small bivalent chromosomes were ring-shaped in the hybrids, respectively (Table 4). The mean number of bivalents per spermatocyte in the controls was 13.00, while that in the hybrids was 12.59 (Table 4). The mature reciprocal hybrids between F. iskandari and F. verruculosa from Indonesia, Lesser Sunda, showed some degree of abnormality in spermatogenesis ( Figure 6).    (Figure 7).

| DISCUSS ION
Speciation, the process by which new species evolve, is a fundamental issue in evolutionary biology and is closely connected to postmating isolation, genetics, and systematics. Mechanisms of postmating (reproductive) isolation are a useful tool to examine the accuracy of species delimitation. It is necessary to categorize sympatric or allopatric species groups (species with homoplasy/phenetic similarities) based on morphological, ecological, and genetic divergence. Wu and Hollocher (1998) reported that an interrelationship between genetic divergence and the degree of reproductive isolation might exist if the number of genes involved in reproductive isolation between taxa is large, with the continuous accumulation of mutations at these loci over time. Similar correlations have been found in Drosophila (Coyne & Orr, 1989), the salamander Desmognathus ochrophaeus (Tilley et al., 1990), and some anurans (Sasa et al., 1998).
However, postmating isolation is not generally observed between all species, and analyses are limited by difficulty in crossing experiments as well as related costs and labor. Despite these barriers, we attempted to cross Fejervarya species from Lesser Sunda, Indonesia, with populations in other Asian countries to quantify the evolutionary relationships and postmating isolation among these frogs.
Reproductive isolation is indispensable for both the generation and preservation of flora and fauna (Dugas & Richards-Zawacki, 2015). The breakdown of reproductive isolation can lead to gene exchange between species, resulting in the sterility or inviability of hybrid offspring (Arnold, 1997). Futuyama (1986)  biologists started to examine reproductive isolation in amphibian in the 1980s. For example, Kawamura et al. (1980) evaluated the postmating isolation mechanism in Japanese, European, and American toads. Later, Kawamura et al. (1981Kawamura et al. ( , 1985 Sumida et al. (2007) and Djong et al. (2007).
Controlled crossing experiments are an essential tool in evolutionary genetics and have applications in population biology, ecology, and phylogenetics (Berger et al., 1994 et al. (2007) and Djong et al. (2007). Postmating isolation between six species belonging to Hoplobatrachus, Euphlyctis, and Fejervarya was also quantified by Alam et al. (2012), with analyses of the degree of abnormality at the genus level, including the production of allotriploids by hybridization. Recently, South Asian frogs were transferred to the genus Minervarya based on observations by several herpetologists (Frost, 2021;Sanchez et al., 2018).
Histological quantification on the testes of hybrids between  Bangladesh, for their concession to collect and transport specimens from Bangladesh to Japan. We are also grateful to anonymous (if any) person/organization who(s) helped to provide the specimens to accomplish this experiment.

CO N FLI C T O F I NTE R E S T
The authors have declared that no competing interests exist.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data related to this article can be accessed here: https://doi.